Vaccine-induced neutralizing antibodies against SARS-CoV-2 Omicron variant isolated in Osaka, Japan

To study vaccine-induced neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants isolated in Osaka, Japan, microneutralization tests were performed on serum samples from 32subjects who received a second dose of vaccination, and 10 of those who received the third dose of vaccination. Geometric mean titres (GMTs) for the D614G strain, Alpha variant, Delta variant, and Omicron BA.1 of the subjects after the second dose of vaccination were 19.5, 21.8, 6.3 and 2.0, respectively. The GMT for the Delta variant was significantly lower than that for the D614G strain and Alpha variant, and the GMT for the Omicron BA.1 was significantly lower than that for the Delta variant. Among the subjects who received three doses of vaccination, the GMTs for the Omicron BA.1 (62.8) and BA.2 (38.6) were significantly higher than that for the Omicron BA.1 after the second dose. Thus, in the present study, the second dose of vaccination induced neutralizing antibodies against SARS-CoV-2 strains, and the reactivity of neutralizing antibodies to the variants was thought to be enhanced by the third dose of vaccination. The serum samples used in this study will be useful in evaluating the reactivity of vaccine-induced antibodies to newly emerging variants.


INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus that causes COVID-19, was first identified at the end of 2019 and has since spread to spawn a worldwide pandemic. SARS-CoV-2 variants have emerged in numerous countries including Japan, which has experienced five waves of the COVID-19 pandemic as of November 2021 [2]. The second and third waves, which began in July and November 2020, respectively, were mainly caused by variants carrying the D614G mutation in the spike protein (B.1.1.214 and B.1.1.284), which enhances infectivity [3]. In the fourth wave, which began in March 2021, the Alpha variant (B.1.1.7) was dominant, especially in Osaka [4]. Meanwhile, the fifth wave, which began in July 2021, was predominated by the highly transmissive Delta variant (B.1.617.2) [5], and the sixth wave, which began in January 2022, was predominated by the highly mutated Omicron variant (B.1.1.519) BA.1 and BA.2 [6].
Vaccines play an important role in the acquisition of immunity to SARS-CoV-2, with several studies having reported their effectiveness against COVID-19 [7,8]. In Japan, vaccinations against COVID-19 started in February 2021, and a third dose is also being administered in 2022 [9]. Serum neutralizing antibody titres can be used to predict vaccine efficacy against SARS-CoV-2 variants of concern [10]. Given that studies on the third dose of vaccine-induced neutralizing antibodies against SARS-CoV-2 variants remain limited in Japan, we used serum samples from vaccinated subjects and measured antibody titres against isolated strains in Osaka using the microneutralization method.

MeTHODS
Blood samples were collected at the Osaka Institute of Public Health for the national epidemiological surveillance of vaccinepreventable diseases. Residual serum from those who had received a second dose of a BNT162b2 mRNA vaccine (Pfizer-BioN-Tech), which encodes the SARS-CoV-2 (Wuhan-Hu-1 strain) full-length spike protein [11], at least three weeks prior were used as samples. In addition, blood samples were collected at least 3 weeks after the third dose of the COVID-19 mRNA vaccine from subjects who provided written informed consent.
For microneutralization tests, serially diluted serum samples starting from a 1 : 5 dilution were mixed with an equal volume of viral solution containing the 50% tissue culture infective dose (100 TCID 50 /50 µl) (final dilution range 1 : 10 to 1 : 1280). The serum-virus mixture was incubated for 1h at 37°C, after which 50µ l of the mixture at each dilution was added to VeroE6/TMPRSS2 cells with 50µl of Dulbecco's Modified Eagle Medium (Sigma, St. Louis, MO, USA) containing 2% fetal bovine serum in 96-well plates. The plates were incubated for 4days at 37°C. Subsequently, the neutralization titre was determined using the endpoint method from the highest dilution at which more than 50 % of cells were protected based on observed cytopathic effects. The microneutralization tests were conducted in duplicate, and for calculation of geometric mean titre (GMT), titre values below 10 were assigned as a value of 1 [13]. Statistical analyses of antibody titres between groups (by variants) were performed using Kruskal-Wallis and Mann-Whitney tests, and Bonferroni adjustment was applied. The correlation between age and antibody titres was evaluated using Spearman's correlation coefficient. Differences in age and gender ratio between two-dose and three-dose vaccination groups were determined by Mann-Whitney test and Fisher's exact test, respectively. SPSS 16.0 software (SPSS, Chicago, IL, USA) was used for the analysis.

ReSUlTS
Serum samples from 32subjects who received two doses of vaccine (age 28 to 67years, mean age 44.0years, percent male 56.3 %) and 10 of those who received three doses of vaccine (age 30 to 64years, mean age 46.7years, percent male 60.0 %) were examined for neutralization of SARS-CoV-2 variants in this study (Table S1, available in the online version of this article). Comparison of the two-dose and three-dose vaccination groups showed no significant differences in age (P=0.44) and gender ratio (P=1). All subjects received the BNT162b2 mRNA COVID-19 vaccine, and blood samples were collected 3-12weeks after the second dose and 3-11 weeks after the third dose. Mutations in the SARS-CoV-2 spike protein of the D614G strain, Alpha, Delta and Omicron variants compared to the vaccine strain are outlined in Table 1 [14,15].
After two doses of vaccination, the GMTs for the D614G strain, Alpha variant, Delta variant and Omicron BA.1 in the subjects were 19.5, 21.8, 6.3 and 2.0, respectively (Fig. 1). There was no statistically significant difference (P=0.36) between the GMTs for the D614G strain and the Alpha variant. In contrast, the GMT for the Delta variant was statistically significantly lower than that for the D614G strain (P <0.0005) and Alpha variant (P <0.00005), and the GMT for the Omicron BA.1 was significantly lower than that for the Delta variant (P <0.005). There was no correlation between age and antibody titre against any of the virus strains with a Spearman correlation coefficient range from −0.077 to −0.036. Likewise, there was no difference in the antibody titre to each strain based on gender (P-value range from 0.42 to 0.89). Fig. 2 shows the antibody titres of the subjects who received the third dose of vaccination. The GMTs for the Omicron BA.1 and BA.2 were 62.8 and 38.6, respectively, and the GMT to BA.2 was lower than that to BA.1, but the difference was not statistically

DISCUSSION
In the present study, we evaluated vaccine-induced antibodies to circulating SARS-CoV-2 variants using a serum neutralization test. Compared to the D614G strain and Alpha variant, the subjects after the second dose of vaccination had lower neutralizing  activity against the Delta variant and Omicron BA.1, which is consistent with reports that immune evasion by the Delta and Omicron variant reduces the sensitivity of vaccine-elicited antibodies [15][16][17]. Unlike the second dose, the third dose of vaccination induced antibodies with neutralizing activity against the Omicron BA.1 and BA.2. These results support previous study regarding the efficacy of booster dose vaccination against the Omicron variant [18]. Although vaccines may not be completely effective for preventing SARS-CoV-2 infection, they are effective against the variants for preventing hospitalization and death [19,20]. It implies that vaccines can stimulate not only humoral immunity but also cellular immunity [21,22].
Since none of the 32subjects who received two doses of vaccine had a history of COVID-19 infection, we assumed that all of the subjects acquired antibodies from vaccination. While previous studies have reported age-and gender-related differences in immune response [23], we could not find the correlation between antibody titre and age or gender. This discrepancy may be due to the limitations of this study, namely the small number of subjects and the inclusion of multiple periods between vaccination and blood collection. Future studies on the seroprevalence of SARS-CoV-2 need to have larger sample sizes and investigate cellular immune responses as well as humoral immune responses.
Our results showed that GMTs against Omicron BA.1 and BA.2 after the third dose increased substantially compared to that after the second dose. The increased reactivity of neutralizing antibodies produced by repeated vaccination against the Omicron variant is thought to be affinity maturation and recall of broadly neutralizing antibodies, as have been reported [24,25]. In this study, antibody titres to Omicron BA.2 were slightly lower than those to BA.1. Differences in neutralizing antibody titres to BA.1 and BA.2 varied among reports, but their differences in antigenicity are considered to be limited [14,17].

CONClUSIONS
The present study demonstrated that the vaccination induced neutralizing antibodies against SARS-CoV-2 strains, and the third dose of vaccination enhanced the reactivity of the neutralizing antibodies to the Omicron variants. Because the effectiveness of the vaccine has been reported to wane over time [26], continuous investigation of antibody levels will be needed. Despite Japan's sixth wave of infection having declined in June 2022, it is important to continue to monitor the recurrence of the Omicron variants including BA.4 and BA.5, and the emergence of new yet-to-be-detected variants. The serum samples used in this study may be useful for evaluating the reactivity of vaccine-induced antibodies to newly emerging variants.

Funding information
This study was supported by Osaka Prefectural Government, and partially supported by KAKENHI (grant number 20K10444) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Author response to reviewers to Version 1
Response to Reviewer 1 We wish to thank you for your valuable comments.We agree with you and have revised our paper in accordance with your comments.
1. The sample size of this paper is very limited and the study only evaluated the neutralization antibody response. There was no attempt to measure the overall antibody response or to examine cell mediated immunity. Realize that this was not the purpose of the study but a large sample size, more frequent sampling of the subjects and more expanded analysis of the immune response would have been a stronger paper.

Response to comments
As you have pointed out, we understand that the sample size of our study is not sufficient. We are considering to increase the number of subjects from whom samples are collected when analyzing immune responses to SARS-CoV-2 in the future.Therefore, we have added the sentence "Future studies on the seroprevalence of SARS-CoV-2 need to have larger sample sizes and investigate cellular immune responses as well as humoral immune responses. " to the limitations of this study. Please see line 144-145of the revised manuscript.
Again, we appreciate all of your insightful comments. Thank you for taking the time to help us improve the paper.
Response to Reviewer 2 We wish to thank you for your valuable comments. We have incorporated your suggestion and revised our paper.

Response to comments
As you suggested, we have revised sentences as "Residual serum from those who had received a second dose of a BNT162b2 mRNA vaccine (Pfizer-BioNTech), which encodes the SARS-CoV-2 (Wuhan-Hu-1 strain) full-length spike protein, at least three weeks prior were used as samples. " in line 66-69.